Polynucleotide separations on polymeric separation media

ABSTRACT

Non-polar polymeric separation media, such as beads or monoliths, are suitable for chromatographic separation of mixtures of polynucleotides when the surfaces of the media are unsubstituted or substituted with a hydrocarbon group having from one to 1,000,000 carbons and when the surfaces are substantially free from mutivalent cation contamination. The polymeric media provide efficient separation of polynucleotides using Matched Ion Polynucleotide Chromatography. Methods for maintaining and storing the polymeric media include treatment with multivalent cation binding agents.

CROSS REFERENCE TO RELATED CO-PENDING APPLICATIONS

This is a continuation-in-part application of U.S. patent applicationSer. No. 09/058,580 filed Apr. 10, 1998 which is hereby incorporated byreference in its entirety. This application is a regular U.S. PatentApplication under 35 U.S.C. §111 (a) and 35 C.F.R. §1.53(b) and claimspriority from the following co-pending, commonly assigned provisionalapplications, each filed under 35 U.S.C. §111(b):

Ser. No. 60/067,679, filed Dec. 5, 1997

Ser. No. 60/077,875, filed Mar. 13, 1998

Ser. No. 60/089,675 filed Jun. 17, 1998

Ser. No. 60/103,313, filed Oct. 6, 1998.

FIELD OF THE INVENTION

The present invention is directed to the separation of polynucleotidesusing non-polar separation surfaces, such as the surfaces of polymericbeads and surfaces within molded monoliths, which are substantially freefrom contamination with multivalent cations.

BACKGROUND OF THE INVENTION

Separations of polynucleotides such as DNA have been traditionallyperformed using slab gel electrophoresis or capillary electrophoresis.However, liquid chromatographic separations of polynucleotides arebecoming more important because of the ability to automate the analysisand to collect fractions after they have been separated. Therefore,columns for polynucleotide separation by liquid chromatography (LC) arebecoming more important.

High quality materials for double stranded DNA separations previouslyhave been based on polymeric substrates disclosed in U.S. Pat. No.5,585,236, to Bonn, et al. (1996), which showed that double-stranded DNAcan be separated on the basis of size with selectivity and performancesimilar to gel electrophoresis using a process characterized as reversephase ion pairing chromatography (RPIPC). However, the chromatographicmaterial described was limited to nonporous beads substituted with alkylgroups having at least 3 carbons because Bonn, et al. were unsuccessfulin obtaining separations using polymer beads lacking this substitution.Additionally, the polymer beads were limited to a small group of vinylaromatic monomers, and Bonn et al. were unable to effect double strandedDNA separations with other materials.

A need continues to exist for chromatographic methods for separatingpolynucleotides with improved separation efficiency and resolution.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide achromatographic method for separating polynucleotides with improvedseparation and efficiency.

Another object of the present invention is to provide a method forseparating polynucleotides using nonporous polymer separation media,such as beads or monoliths (e.g., rods), having non-reactive, non-polarsurfaces.

It is another object of this invention to provide the chromatographicseparation of polynucleotides using nonporous polymeric separation mediamade from a variety of different polymerizable monomers.

It is a further object of this invention to provide the chromatographicseparation of polynucleotides using polymeric separation media which canbe unsubstituted, methyl-substituted, ethyl-substituted,hydrocarbon-substituted, or hydrocarbon polymer-substituted.

Yet another object of the p resent invention is to provide improvedpolymer separation media by including steps to remove contaminationoccurring during the manufacturing process.

Still another object of the invention is to provide a method forseparating polynucleotides using a variety of different solvent systems.

These and other objects which will become apparent from the followingspecification have been achieved by the present invention.

In one aspect, the invention is a method for separating a mixture ofpolynucleotides by applying a mixture of polynucleotides having up to1500 base pairs to a polymeric separation medium having non-polarsurfaces which are substantially free from contamination withmultivalent cations, and eluting the mixture of polynucleotides. Thepreferred surfaces are nonporous. The non-polar surfaces can be enclosedin a column. In the preferred embodiment, precautions are taken duringthe production of the medium so that it is substantially free ofmultivalent cation contaminants and the medium is treated, for exampleby an acid wash treatment and/or treatment with multivalent cationbinding agent, to remove any residual surface metal contaminants. Thepreferred separation medium is characterized by having a DNA SeparationFactor (defined hereinbelow) of at least 0.05. The preferred separationmedium is also characterized by having a Mutation Separation Factor (asdefined hereinbelow) of at least 0.1. In the preferred embodiment, theseparation is made by Matched Ion Polynucleotide Chromatography (MIPC,as defined hereinbelow). Examples of non-polar surfaces include thesurfaces of polymer beads and the surfaces of interstitial spaces withina polymeric monolith. The elution step preferably uses a mobile phasecontaining a counterion agent and a water-soluble organic solvent.Examples of a suitable organic solvent include alcohol, nitrile,dimethylformamide, tetrahydrofuran, ester, ether, and mixtures of one ormore thereof, e.g., methanol, ethanol, 2-propanol, 1-propanol,tetrahydrofuran, ethyl acetate, acetonitrile. The most preferred organicsolvent is acetonitrile. The counterion agent is preferably selectedfrom the group consisting of lower alkyl primary amine, lower alkylsecondary amine, lower alkyl tertiary amine, lower trialkyammonium salt,quaternary ammonium salt, and mixtures of one or more thereof.Non-limiting examples of counterion agents include octylammoniumacetate, octadimethylammonium acetate, decylammonium acetate,octadecylammonium acetate, pyridiniumammonium acetate,cyclohexylammonium acetate, diethylammonium acetate, propylethylammoniumacetate, propyldiethylammonium acetate, butylethylammonium acetate,methylhexylammonium acetate, etramethylammonium acetate,tetrapropylammonium acetate, etrabutylammonium acetate,dimethydiethylammon ium acetate, triethylammonium acetate,tripropylammonium acetate, tributylammonium acetate, tetraethylammoniumacetate, tetrapropylammonium acetate, tetrabutylammonium acetate, andmixtures of any one or more of the above. The counterion agent includesan anion, e.g., acetate, carbonate, bicarbonate, phosphate, sulfate,nitrate, propionate, formate, chloride, perchlorate, or bromide. Themost preferred counterion agent is triethylammonium acetate ortriethylammonium hexafluoroisopropyl alcohol.

One embodiment of the invention provides a method for separating amixture of polynucleotides, comprising applying a mixture ofpolynucleotides having up to 1500 base pairs to polymeric separationbeads having non-polar surfaces which are substantially free fromcontamination with multivalent cations, and eluting said mixture ofpolynucleotides. In a particular embodiment of the separation medium,the invention provides a method for separating a mixture ofpolynucleotides comprising flowing a mixture of polynucleotides havingup to 1500 base pairs through a separation column containing polymerbeads which are substantially free from contamination with multivalentcations and having an average diameter of 0.5 to 100 microns, andseparating the mixture of polynucleotides. The beads are preferably madefrom polymers, including mono- and di-vinyl substituted aromaticcompounds such as styrene, substituted styrenes, alpha-substitutedstyrenes and divinylbenzene; acrylates and methacrylates; polyolefinssuch as polypropylene and polyethylene; polyesters; polyurethanes;polyamides; olycarbonates; and substituted polymers includingfluorosubstituted thylenes commonly known under the trademark TEFLON.The base polymer can also be mixtures of polymers, non-limiting examplesof which include poly(styrene-divinylbenzene) andpoly(ethylvinylbenzene-divinylbenzene). The polymer can beunsubstituted, or substituted with a hydrocarbon such as an alkyl grouphaving from 1 to 1,000,000 carbons. In a preferred embodiment, thehydrocarbon is an alkyl group having from 1 to 24 carbons. In morepreferred embodiment, the alkyl group has 1-8 carbons. The beadspreferably have an average diameter of about 1-5 microns. In thepreferred embodiment, precautions are taken during the production of thebeads so that they are substantially free of multivalent cationcontaminants and the beads are treated, for example by an acid washtreatment, to remove any residual surface metal contaminants. The beadsof the invention are characterized by having a DNA Separation Factor ofat least 0.05. In a preferred embodiment, the beads are characterized byhaving a DNA Separation Factor of at least 0.5. Also in a preferredembodiment, the beads are characterized by having a Mutation SeparationFactor of at least 0.1. The preferred method used in the separation ismade by MIPC. In one embodiment, the beads are used in a capillarycolumn to separate a mixture of polynucleotides by capillaryelectrochromatography. In other embodiments, the beads are used toseparate the mixture by thin-layer chromatography or by high-speedthin-layer chromatography.

In addition to the beads (or other media) themselves being substantiallymetal-free, Applicants have also found that to achieve optimum peakseparation the inner surfaces of the separation column (or othercontainer) and all process solutions held within the column or flowingthrough the column are preferably substantially free of multivalentcation contaminants. This can be achieved by supplying and feedingsolutions entering the separation column with components which haveprocess solution-contacting surfaces made of material which does notrelease multivalent cations into the process solutions held within orflowing through the column, in order to protect the column frommultivalent cation contamination. The process solution-contactingsurfaces of the system components are preferably material selected fromthe group consisting of titanium, coated stainless steel, and organicpolymer.

For additional protection, multivalent cations in mobile phase solutionsand sample solutions entering the column can be removed by contactingthese solutions with multivalent cation capture resin before thesolutions enter the column to protect the separation medium frommultivalent cation contamination. The multivalent capture resin ispreferably cation exchange resin and/or chelating resin. The method ofthe present invention can be used to separate double strandedpolynucleotides having up to about 1500 to 2000 base pairs. In manycases, the method is used to separate polynucleotides having up to 600bases or base pairs, or which have up to 5 to 80 bases or base pairs.The mixture of polynucleotides can be a polymerase chain reactionproduct. The method preferably is performed at a temperature within therange of 20° C. to 90° C. The flow rate of mobile phase preferably isadjusted to yield a back-pressure not greater than 5000 psi. The methodreferably employs an organic solvent that is water soluble. The methodalso referably employs a counterion agent.

In another aspect, the present invention provides a polymeric beadhaving an average bead diameter of 0.5-100 micron. Precautions are takenduring the production of the beads so that they are substantially freeof multivalent cation contaminants and the beads are treated, forexample by an acid wash treatment, to remove any residual surface metalcontaminants. In one embodiment, the beads are characterized by having aDNA Separation Factor of at least 0.05. In a preferred embodiment, thebeads are characterized by having a DNA Separation Factor of at least0.5. In a preferred embodiment, the beads are characterized by having aMutation Separation Factor of at least 0.1. The bead preferably has anaverage diameter of about 1-10 microns, and most preferably has anaverage diameter of about 1-5 microns. The bead can be comprised of acopolymer of vinyl aromatic monomers. The vinyl aromatic monomers can bestyrene, alkyl substituted styrene, alpha-methylstyrene or alkylsubstituted alpha-methylstyrene. The bead can be a copolymer such as acopolymer of styrene, C₁₋₆ alkyl vinylbenzene and divinylbenzene. Thebead can contain functional groups such as polyvinyl alcohol, hydroxy,nitro, halogen (e.g. bromo), cyano, aldehyde, or other groups that donot bind the sample. The bead can be unsubstituted or having boundthereto a hydrocarbon group having from 1 to 1,000,000 carbons. In oneembodiment, the hydrocarbon group is an alkyl group having from 1 to 24carbons. In another embodiment, the hydrocarbon group has from 1 to 8carbons. In preferred embodiments, he bead is octadecyl modifiedpoly(ethylvinylbenzene-divinylbenzene) or poly(styrene-divinylbenzene).The bead can also contain crosslinking divinylmonomer such as divinylbenzene or butadiene.

In yet another embodiment, the invention is a method for separating amixture of polynucleotides comprising flowing a mixture ofpolynucleotides having up to 1500 base pairs through a polymericmonolith, and separating the mixture of polynucleotides using MIPC. Inthis embodiment, the non-polar separation surfaces are the surfaces ofinterstitial spaces of a polymeric monolith. An example of such amonolith is a polymeric rod prepared within the confines of achromatographic column. The monolith of the invention is characterizedby having a DNA Separation Factor of at least 0.05. In a preferredembodiment, the monolith is characterized by having a DNA SeparationFactor of at least 0.5. The monolith is preferably characterized byhaving a Mutation Separation Factor of at least 0.1. The mobile phaseused in the separation preferably includes an organic solvent asexemplified by alcohol, nitrile, dimethylformamide, tetrahydrofuran,ester, ether, and mixtures thereof. Examples of suitable solventsinclude methanol, ethanol, 2-propanol, 1-propanol, tetrahydrofuran,ethyl acetate, acetonitrile, and mixtures thereof. The most preferredorganic solvent is acetonitrile. The mobile phase preferably includes acounterion agent such as lower primary, secondary and tertiary amines,and lower trialkyammonium salts, or quaternary ammonium salts. Morespecifically, the counterion agent can be octylammonium acetate,octadimethylammonium acetate, decylammonium acetate, octadecylammoniumacetate, pyridiniumammonium acetate, cyclohexylammonium acetate,diethylammonium acetate, propylethylammonium acetate,propyidiethylammonium acetate, butylethylammonium acetate,methylhexylammonium acetate, tetramethylammonium acetate,tetraethylammonium acetate, tetrapropylammonium acetate,tetrabutylammonium acetate, dimethydiethylammonium acetate,triethylammonium acetate, tripropylammonium acetate, tributylammoniumacetate, tetrapropylammonium acetate, tetrabutylammonium acetate, andmixtures of any one or more of the above. The counterion agent includesan anion, e.g., acetate, carbonate, bicarbonate, phosphate, sulfate,nitrate, propionate, formate, chloride, perchlorate, and bromide.However, the most preferred counterion agent is triethylammoniumacetate.

In the preferred embodiment, precautions are taken during the productionof the polymeric monolith so that it is substantially free ofmultivalent cation contaminants and the monolith is treated, forexample, by an acid wash treatment, to remove any residual surface metalcontaminants. In one embodiment, the monolith is characterized by havinga DNA Separation Factor of at least 0.05. In a preferred embodiment, themonolith is characterized by having a DNA Separation Factor of at least0.5. Also in a preferred embodiment, the monolith is characterized byhaving a Mutation Separation Factor of at least 0.1.

In another aspect, the present invention is a method for treating thenon-polar surface of a polymeric medium used for separatingpolynucleotides, such as the surface of beads in a MIPC column or theinterstitial spaces in a polymeric monolith, in order to improve theresolution of polynucleotides, such as dsDNA, separated on said surface.This treatment includes contacting the surface with a solutioncontaining a multivalent cation binding agent. In a preferredembodiment, the solution has a temperature of about 50° C. to 90° C. Anexample of this treatment includes flowing a solution containing amultivalent cation binding agent through a MIPC column, wherein thesolution has a temperature of about 50° C. to 90° C. The preferredtemperature is about 70° C. to 80° C. In a preferred embodiment, themultivalent cation binding agent is a coordination compound, examples ofwhich include water-soluble chelating agents and crown ethers. Specificexamples include acetylacetone, alizarin, aluminon, chloranilic acid,kojic acid, morin, rhodizonic acid, thionalide, thiourea,α-furildioxime, nioxime, salicylaldoxime, dimethylglyoxime,α-furildioxime, cupferron, α-nitroso-β-naphthol, nitroso-R-salt,diphenylthiocarbazone, diphenylcarbazone, eriochrome black T, PAN,SPADNS, glyoxal-bis(2-hydroxyanil), murexide, α-benzoinoxime, mandelicacid, anthranilic acid, ethylenediamine, glycine, triaminotriethylamine,thionalide, triethylenetetramine, ethylenediaminetetraacetic acid(EDTA), metalphthalein, arsonic acids, α,α'-bipyridine,4-hydroxybenzothiazole, 8-hydroxyquinaldine, 8-hydroxyquinoline,1,10-phenanthroline, picolinic acid, quinaldic acid, α,α',α"-terpyridyl,9-methyl-2,3,7-trihydroxy-6-fluorone, pyrocatechol, salicylic acid,tiron, 4-chloro-1,2-dimercaptobenzene, dithiol, mercaptobenzothiazole,rubeanic acid, oxalic acid, sodium diethyldithiocarbarbamate, and zincdibenzyldithiocarbamate. However, the most preferred chelating agent isEDTA. In this aspect of the invention, the solution preferably includesan organic solvent as exemplified by alcohol, nitrile,dimethylformamide, tetrahydrofuran, ester, ether, and mixtures thereof.Examples of suitable solvents include methanol, ethanol, 2-propanol,1-propanol, tetrahydrofuran, ethyl acetate, acetonitrile, and mixturesthereof. The most preferred organic solvent is acetonitrile. In oneembodiment, the solution can include a counterion agent such as lowerprimary, secondary and tertiary amines, and lower trialkyammonium salts,or quaternary ammonium salts. More specifically, the counterion agentcan be octylammonium acetate, octadimethylammonium acetate,decylammonium acetate, octadecylammonium acetate, pyridiniumammoniumacetate, cyclohexylammonium acetate, diethylammonium acetate,propylethylammonium acetate, propyldiethylammonium acetate,butylethylammonium acetate, methyihexylammonium acetate,tetramethylammonium acetate, tetraethylammonium acetate,tetrapropylammonium acetate, tetrabutylammonium acetate,dimethydiethylammonium acetate, triethylammonium acetate,tripropylammonium acetate, tributylammonium acetate, tetrapropylammoniumacetate, tetrabutylammonium acetate, and mixtures of any one or more ofthe above. The counterion agent includes an anion, e.g., acetate,carbonate, bicarbonate, phosphate, sulfate, nitrate, propionate,formate, chloride, perchlorate, and bromide. However, the most preferredcounterion agent is riethylammonium acetate.

In yet a further aspect, the invention provides a method for storing amedium used for separating polynucleotides, e.g., the beads of a MIPCcolumn or a polymeric monolith, in order to improve the resolution ofdouble stranded DNA fragments separated using the medium. In the case ofa MIPC column, the preferred method includes flowing a solutioncontaining a multivalent cation binding agent through the column priorto storing the column. In a preferred embodiment, the multivalent cationbinding agent is a coordination compound, examples of which includewater-soluble chelating agents and crown ethers. Specific examplesinclude acetylacetone, alizarin, aluminon, chloranilic acid, kojic acid,morin, rhodizonic acid, thionalide, thiourea, α-furildioxime, nioxime,salicylaldoxime, dimethylglyoxime, α-furildioxime, cupferron,α-nitroso-β-naphthol, nitroso-R-salt, diphenylthiocarbazone,diphenylcarbazone, eriochrome black T, PAN, SPADNS,glyoxal-bis(2-hydroxyanil), murexide, α-benzoinoxime, mandelic acid,anthranilic acid, ethylenediamine, glycine, triaminotriethylamine,thionalide, triethylenetetramine, EDTA, metalphthalein, arsonic acids,α,α'-bipyridine, 4-hydroxybenzothiazole, 8-hydroxyquinaldine,8-hydroxyquinoline, 1,10-phenanthroline, picolinic acid, quinaldic acid,α,α',α"-terpyridyl, 9-methyl-2,3,7-trihydroxy-6-fluorone, pyrocatechol,salicylic acid, tiron, 4-chloro-1,2-dimercaptobenzene, dithiol,mercaptobenzothiazole, rubeanic acid, oxalic acid, sodiumdiethyldithiocarbarbamate, and zinc dibenzyldithiocarbamate. However,the most preferred chelating agent is EDTA. In this aspect of theinvention, the solution preferably includes an organic solvent asexemplified by alcohols, nitriles, dimethylformamide, tetrahydrofuran,esters, and ethers. The most preferred organic solvent is acetonitrile.The solution can also include a counterion agent such as lower primary,secondary and tertiary amines, and lower trialkyammonium salts, orquaternary ammonium salts. More specifically, the counterion agent canbe octylammonium acetate, octadimethylammonium acetate, decylammoniumacetate, octadecylammonium acetate, pyridiniumammonium acetate,cyclohexylammonium acetate, diethylammonium acetate, propylethylammoniumacetate, propyidiethylammonium acetate, butylethylammonium acetate,methylhexylammonium acetate, tetramethylammonium acetate,tetraethylammonium acetate, tetrapropylammonium acetate,tetrabutylammonium acetate, dimethydiethylammonium acetate,triethylammonium acetate, tripropylammonium acetate, tributylammoniumacetate, tetrapropylammonium acetate, tetrabutylammonium acetate, andmixtures of any one or more of the above. The counterion agent includesan anion, e.g., acetate, carbonate, bicarbonate, phosphate, sulfate,nitrate, propionate, formate, chloride, perchlorate, and bromide.However, the most preferred counterion agent is triethylammoniumacetate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of how the DNA Separation Factor ismeasured.

FIG. 2 is a MIPC separation of pUC18 DNA-HaeIII digestion fragments on acolumn containing alkylated poly(styrene-divinylbenzene) beads. Peaksare labeled with the number of base pairs of the eluted fragment.

FIG. 3 is a MIPC separation of pUC18 DNA-HaeIII digestion fragments on acolumn containing nonporous 2.1 micron beads of underivatizedpoly(styrene-divinylbenzene).

FIG. 4 is a Van't Hoff plot of Ink vs. 1/T [^(o) K⁻¹ ] with alkylatedpoly(styrene-divinylbenzene) beads showing positive enthalpy usingacetonitrile as the solvent.

FIG. 5 is a Van't Hoff plot of In k vs. 1/T [^(o) K⁻¹ ] withunderivatized poly(styrene-divinylbenzene) beads showing positiveenthalpy using acetonitrile as the solvent.

FIG. 6 is a Van't Hoff plot of In k vs. 1/T [^(o) K⁻¹ ] with alkylatedpoly(styrene-divinylbenzene) beads showing negative enthalpy usingmethanol as the solvent.

FIG. 7 is a separation using alkylated beads and acetonitrile assolvent.

FIG. 8 is a separation using alkylated beads and 50.0% methanol as thesolvent.

FIG. 9 is a separation using alkylated beads and 25.0% ethanol as thesolvent.

FIG. 10 is a separation using alkylated beads and 25.0% vodka (100proof) as the solvent.

FIG. 11 is a separation using alkylated beads and 25.0% 1 -propanol asthe solvent.

FIG. 12 is a separation using alkylated beads and 25.0% 1-propanol asthe solvent.

FIG. 13 is a separation using alkylated beads and 10.0% 2-propanol asthe solvent.

FIG. 14 is a separation using alkylated beads and 10.0% 2-propanol asthe solvent.

FIG. 15 is a separation using alkylated beads and 25.0% THF as thesolvent.

FIG. 16 is a combination isocratic/gradient separation on anon-alkylated poly(styrene-divinylbenzene) beads.

FIG. 17 shows a schematic representation of a hybridization to formhomoduplex and heteroduplex.

FIG. 18 is an elution profile showing separation of a 209 base pairhomoduplex/heteroduplex mutation detection mixture performed by DMIPC at56° C.

FIG. 19 is an elution profile of another injection of the same 209 bpmixture and using the same column as in FIG. 18, but after changing theguard cartridge and replacing the pump-valve filter.

FIG. 20 is an elution profile of another injection of the same 209 bpmixture and using the same column as in FIG. 19, but after flushing thecolumn with 0.1 M TEAA, 25% acetonitrile, and 0.32 M EDTA for 45 minutesat 75° C.

FIG. 21 is a DMIPC elution profile of a 100 bp PCR product from awild-type strand of Lambda DNA.

FIG. 22 is a DMIPC elution profile of a hybridized mixture containing aLambda DNA strand containing a mutation and wild type strand.

DETAILED DESCRIPTION OF THE INVENTION

In its most general form, the subject matter of the present inventionconcerns the separation of polynucleotides. e.g. DNA, utilizing astationary separation medium having non-polar surfaces. The preferredsurfaces are essentially free from multivalent cation contaminationwhich can trap polynucleotides. The separation is performed on thestationary surface. The surface can be porous, but preferably anysurface pores are of a size which excludes the smallest polynucleotidebeing analyzed.

The medium can be enclosed in a column. In one embodiment, the non-polarsurfaces comprise the surfaces of polymeric beads. In an alternativeembodiment, the surfaces comprise the surfaces of interstitial spaces ina molded polymeric monolith. For purposes of simplifying the descriptionof the invention and not by way of limitation, the separation ofpolynucleotides using nonporous beads, and the preparation of suchbeads, will be primarily described herein, it being understood thatother separation surfaces, such as the interstitial surfaces ofpolymeric monoliths, are intended to be included within the scope ofthis invention. Monoliths such as rods contain polymer separation mediawhich have been formed inside a column as a unitary structure havingthrough pores or interstitial spaces which allow eluting solvent andanalyte to pass through and which provide the non-polar separationsurface.

In general, the only requirement for the separation media of the resentinvention is that they must have a surface that is either intrinsicallynon-polar or be bonded with a material that forms a surface havingsufficient non-polarity to interact with a counterion agent.

In one aspect, the subject matter of the present invention is theseparation of polynucleotides utilizing columns filled with nonporouspolymeric beads having an average diameter of about 0.5-100 microns;preferably, 1-10 microns; more preferably, 1-5 microns. Beads having anaverage diameter of 1.0-3.0 microns are most preferred.

In U.S. Pat. No. 5,585,236, Bonn et al. had characterized the nucleicacid separation process as reverse phase ion pairing chromatography(RPIPC). However, since RPIPC does not incorporate certain essentialcharacteristics described in the present invention, another term,Matched Ion Polynucleotide Chromatography (MIPC), has been selected.MIPC as used herein, is defined as a process for separating single anddouble stranded polynucleotides using non-polar beads, wherein theprocess uses a counterion agent, and an organic solvent to elute thenucleic acid from the beads, and wherein the beads are characterized ashaving a DNA Separation Factor of at least 0.05. In a preferredembodiment, the beads have a DNA Separation Factor of at least 0.5. Inan optimal embodiment, the beads have a DNA Separation Factor of atleast 0.95.

The performance of the beads of the present invention is demonstrated byhigh efficiency separation by MIPC of double stranded and singlestranded DNA. Applicants have found that a useful criterion formeasuring performance of the beads is a DNA Separation Factor. This ismeasured as he resolution of 257- and 267-base pair double stranded DNAfragments of a pUC18 DNA-HaeIII restriction digest and is defined as theratio of the distance from the valley between the peaks to the top ofthe peaks, over the distance from the baseline to the top of the peaks.Referring to the schematic representation of FIG. 1, the DNA SeparationFactor is determined by measuring the distance "a" from the baseline tothe valley "e" between the peaks "b" and "c" and the distance "d" fromthe valley "e" to the top of one of the peaks "b" or "c". If the peakheights are unequal, the highest peak is used to obtain "d." The DNASeparation Factor is the ratio of d/(a+d). The peaks of 257- and267-base pairs in this schematic representation are similar in height.Operational beads of the present invention have a DNA Separation Factorof at least 0.05. Preferred beads have a DNA Separation Factor of atleast 0.5.

Without wishing to be bound by theory, Applicants believe that the beadswhich conform to the DNA Separation Factor as specified herein have apore size which essentially excludes the polynucleotides being separatedfrom entering the bead. As used herein, the term "nonporous" is definedto denote a bead which has surface pores having a diameter that is lessthan the size and shape of the smallest DNA fragment in the separationin the solvent medium used therein. Included in this definition arepolymer beads having these specified maximum size restrictions in theirnatural state or which have been treated to reduce their pore size tomeet the maximum effective pore size required. Preferably, all beadswhich provide a DNA Separation Factor of at least 0.5 are intended to beincluded within the definition of "nonporous" beads.

The surface conformations of nonporous beads of the present inventioncan include depressions and shallow pit-like structures which do notinterfere with the separation process. A pretreatment of a porous beadto render it nonporous can be effected with any material which will fillthe pores in the bead structure and which does not significantlyinterfere with the MIPC process.

Pores are open structures through which mobile phase and other materialscan enter the bead structure. Pores are often interconnected so thatfluid entering one pore can exit from another pore. Applicants believethat pores having dimensions that allow movement of the polynucleotideinto the interconnected pore structure and into the bead impair theresolution of separations or result in separations that have very longretention times. In MIPC, however, the beads are "nonporous" and thepolynucleotides do not enter the bead structure.

The term polynucleotide is defined as a linear polymer containing anindefinite number of nucleotides, linked from one ribose (ordeoxyribose) to another via phosphoric residues. The present inventioncan be used in the separation of RNA or of double- or single-strandedDNA. For purposes of simplifying the description of the invention, andnot by way of limitation, the separation of double-stranded DNA will bedescribed in the examples herein, it being understood that allpolynucleotides are intended to be included within the scope of thisinvention.

Chromatographic efficiency of the column beads is predominantlyinfluenced by the properties of surface and near-surface areas. For thisreason, the following descriptions are related specifically to theclose-to-the-surface region of the polymeric beads. The main body and/orthe center of such beads can exhibit entirely different chemistries andsets of physical properties from those observed at or near the surfaceof the polymeric beads of the present invention.

In another embodiment of the present invention, the separation mediumcan be in the form of a polymeric monolith such as a rod-like monolithiccolumn. The monolithic column is polymerized or formed as a single unitinside of a tube as described in the Examples hereinbelow. The throughpore or interstitial spaces provide for the passage of eluting solventand analyte materials. The separation is performed on the stationarysurface. The surface can be porous, but is preferably nonporous. Theform and function of the separations are identical to columns packedwith beads. As with beads, the pores contained in the rod must becompatible with DNA and not trap the material. Also, the rod must notcontain contamination that will trap DNA.

The molded polymeric rod of the present invention is prepared by bulkfree radical polymerization within the confines of a chromatographiccolumn. The base polymer of the rod can be produced from a variety ofpolymerizable monomers. For example, the monolithic rod can be made frompolymers, including mono- and di-vinyl substituted aromatic compoundssuch as styrene, substituted styrenes, alpha-substituted styrenes anddivinylbenzene; acrylates and methacrylates; polyolefins such aspolypropylene and polyethylene; polyesters; polyurethanes; polyamides;polycarbonates; and substituted polymers including fluorosubstitutedethylenes commonly known under the trademark TEFLON. The base polymercan also be mixtures of polymers, non-limiting examples of which includepoly(glycidyl methacrylate-co-ethylene dimethacrylate),poly(styrene-divinylbenzene) and poly(ethylvinylbenzene-divinylbenzene.The rod can be unsubsituted or substituted with a substituent such as ahydrocarbon alkyl or an aryl group. The alkyl group optionally has 1 to1,000,000 carbons inclusive in a straight or branched chain, andincludes straight chained, branch chained, cyclic, saturated,unsaturated nonionic functional groups of various types includingaldehyde, ketone, ester, ether, alkyl groups, and the like, and the arylgroups includes as monocyclic, bicyclic, and tricyclic aromatichydrocarbon groups including phenyl, naphthyl, and the like. In apreferred embodiment, the alkyl group has 1-24 carbons. In a morepreferred embodiment, the alkyl group has 1-8 carbons. The substitutioncan also contain hydroxy, cyano, nitro groups, or the like which areconsidered to be non-polar, reverse phase functional groups. Methods forhydrocarbon substitution are conventional and well-known in the art andare not an aspect of this invention. The preparation of polymericmonoliths is by conventional methods well known in the art as describedin the following references: Wang et al. (J. Chromatog. A 699:230(1994)), Petro et al. (Ana. Chem. 68:315 (1996)), and the following U.S.Pat. Nos. 5,334,310; 5,453,185; 5,522,994 (to Frechet). Monolith or rodcolumns are commercially available form Merck & Co (Darmstadt, Germany).

The nonporous polymeric beads of the present invention are prepared by atwo-step process in which small seed beads are initially produced byemulsion polymerization of suitable polymerizable monomers. The emulsionpolymerization procedure of the invention is a modification of theprocedure of Goodwin, et al. (Colloid & Polymer Sci., 252:464-471(1974)). Monomers which can be used in the emulsion polymerizationprocess to produce the seed beads include styrene, alkyl substitutedstyrenes, alpha-methyl styrene, and alkyl substituted alpha-methylstyrene. The seed beads are then enlarged and, optionally, modified bysubstitution with various groups to produce the nonporous polymericbeads of the present invention.

The seed beads produced by emulsion polymerization can be enlarged byany known process for increasing the size of the polymer beads. Forexample, polymer beads can be enlarged by the activated swelling processdisclosed in U.S. Pat. No. 4,563,510. The enlarged or swollen polymerbeads are further swollen with a crosslinking polymerizable monomer anda polymerization initiator. Polymerization increases the crosslinkingdensity of the enlarged polymeric bead and reduces the surface porosityof the bead. Suitable crosslinking monomers contain at least twocarbon-carbon double bonds capable of polymerization in the presence ofan initiator. Preferred crosslinking monomers are divinyl monomers,preferably alkyl and aryl (phenyl, naphthyl, etc.) divinyl monomers andinclude divinyl benzene, butadiene, etc. Activated swelling of thepolymeric seed beads is useful to produce polymer beads having anaverage diameter ranging from 1 up to about 100 microns.

Alternatively, the polymer seed beads can be enlarged simply by heatingthe seed latex resulting from emulsion polymerization. This alternativeeliminates the need for activated swelling of the seed beads with anactivating solvent. Instead, the seed latex is mixed with thecrosslinking monomer and polymerization initiator described above,together with or without a water-miscible solvent for the crosslinkingmonomer. Suitable solvents include acetone, tetrahydrofuran (THF),methanol, and dioxane. The resulting mixture is heated for about 1-12hours, preferably about 4-8 hours, at a temperature below the initiationtemperature of the polymerization initiator, generally, about 10° C.-80°C., preferably 30° C.-60° C. Optionally, the temperature of the mixturecan be increased by 10-20% and the mixture heated for an additional 1 to4 hours. The ratio of monomer to polymerization initiator is at least100:1, preferably about 100:1 to about 500:1, more preferably about200:1 in order to ensure a degree of polymerization of at least 200.Beads having this degree of polymerization are sufficientlypressure-stable to be used in high pressure liquid chromatography (HPLC)applications. This thermal swelling process allows one to increase thesize of the bead by about 110-160% to obtain polymer beads having anaverage diameter up to about 5 microns, preferably about 2-3 microns.The thermal swelling procedure can, therefore, be used to producesmaller particle sizes previously accessible only by the activatedswelling procedure.

Following thermal enlargement, excess crosslinking monomer is removedand the particles are polymerized by exposure to ultraviolet light orheat. Polymerization can be conducted, for example, by heating of theenlarged particles to the activation temperature of the polymerizationinitiator and continuing polymerization until the desired degree ofpolymerization has been achieved. Continued heating and polymerizationallows one to obtain beads having a degree of polymerization greaterthan 500.

In the present invention, the packing material disclosed by Bonn et al.or U.S. Pat. No. 4,563,510 can be modified through substitution of thepolymeric beads with alkyl groups or can be used in its unmodifiedstate. For example, the polymer beads can be alkylated with 1 or 2carbon atoms by contacting the beads with an alkylating agent, such asmethyl iodide or ethyl iodide. Alkylation is achieved by mixing thepolymer beads with the alkyl halide in the presence of a Friedel-Craftscatalyst to effect electrophilic aromatic substitution on the aromaticrings at the surface of the polymer blend. Suitable Friedel-Craftscatalysts are well-known in the art and include Lewis acids such asaluminum chloride, boron trifluoride, tin tetrachloride, etc. The beadscan be hydrocarbon substituted by substituting the correspondinghydrocarbon halide for methyl iodide in the above procedure, forexample.

The term alkyl as used herein in reference to the beads of the presentinvention is defined to include alkyl and alkyl substituted aryl groups,having from 1 to 1,000,000 carbons, the alkyl groups including straightchained, branch chained, cyclic, saturated, unsaturated nonionicfunctional groups of various types including aldehyde, ketone, ester,ether, alkyl groups, and the like, and the aryl groups including asmonocyclic, bicyclic, and tricyclic aromatic hydrocarbon groupsincluding phenyl, naphthyl, and the like. Methods for alkyl substitutionare conventional and well-known in the art and are not an aspect of thisinvention. The substitution can also contain hydroxy, cyano, nitrogroups, or the like which are considered to be non-polar, reverse phasefunctional groups.

The chromatographic material reported in the Bonn patent was limited tononporous beads substituted with alkyl groups having at least 3 carbonsbecause Bonn et al. were unsuccessful in obtaining separations usingpolymer beads lacking this substitution. Additionally, the polymer beadswere limited to a small group of vinyl aromatic monomers, and Bonn etal. were unable to effect double stranded DNA separations with othermaterials.

In the present invention, it has now been surprisingly discovered thatsuccessful separation of double stranded DNA can be achieved usingunderivatized nonporous beads as well as using beads derivatized withalkyl groups having 1 to 1,000,000 carbons.

The polymer is preferably unsubstituted or substituted with methyl,ethyl, or hydrocarbon having from 23 to 1,000,000 carbons.

The base polymer of the invention can also be other polymers,non-limiting examples of which include mono- and di-vinyl substitutedaromatics such as styrene, substituted styrenes, alpha-substitutedstyrenes and divinylbenzene; acrylates and methacrylates; polyolefinssuch as polypropylene and polyethylene; polyesters; polyurethanes;polyamides; polycarbonates; and substituted polymers includingfluorosubstituted ethylenes commonly known under the trademark TEFLON.The base polymer can also be mixtures of polymers, non-limiting examplesof which include poly(styrene-divinylbenzene) andpoly(ethylvinylbenzene-divinylbenzene). Methods for making beads fromthese polymers are conventional and well known in the art (for example,see U.S. Pat. No. 4,906,378). The physical properties of the surface andnear-surface areas of the beads are the predominant influence onchromatographic efficiency. The polymer, whether derivatized or not,must provide a nonporous, non-reactive, and non-polar surface for theMIPC separation.

In an important aspect of the present invention, the beads and othermedia of the invention are characterized by having low amounts of metalcontaminants or other contaminants that can bind DNA. The preferredbeads of the present invention are characterized by having beensubjected to precautions during production, including a decontaminationtreatment, such as an acid wash treatment, designed to substantiallyeliminate any multivalent cation contaminants (e.g. Fe(III), Cr(III), orcolloidal metal contaminants). Only very pure, non-metal containingmaterials should be used in the production of the beads in order thatthe resulting beads will have minimum metal content.

In addition to the beads themselves being substantially metal-free,Applicants have also found that, to achieve optimum peak separationduring MIPC, the separation column and all process solutions held withinthe column or flowing through the column are preferably substantiallyfree of multivalent cation contaminants. As described in commonly ownedU.S. Pat. No. 5,772,889 to Gjerde (1998), and in co-pending U.S. patentapplications Ser. No. 09/081,040 (filed May 18, 1998) and No. 09/080,547(filed May 18, 1998) this can be achieved by supplying and feedingsolutions that enter the separation column with components which haveprocess solution-contacting surfaces made of material which does notrelease multivalent cations into the process solutions held within orflowing through the column, in order to protect the column frommultivalent cation contamination. The process solution-contactingsurfaces of the system components are preferably material selected fromthe group consisting of titanium, coated stainless steel, passivatedstainless steel, and organic polymer.

There are two places where multivalent cation binding agents, e.g.,chelators, are used in MIPC separations. In one embodiment, thesebinding agents can be incorporated into a solid through which the mobilephase passes. Contaminants are trapped before they reach places withinthe system that can harm the separation. In these cases, the functionalgroup is attached to a solid matrix or resin (e.g., a flow-throughcartridge, usually an organic polymer, but sometimes silica or othermaterial). The capacity of the matrix is preferably about 2 mequiv./g.An example of a suitable chelating resin is available under thetrademark CHELEX 100 (Dow Chemical Co.) containing an iminodiacetatefunctional group.

In another embodiment, the multivalent cation binding agent can be addedto the mobile phase. The binding functional group is incorporated intoan organic chemical structure. The preferred multivalent cation bindingagent fulfills three requirements. First, it is soluble in the mobilephase. Second, the complex with the metal is soluble in the mobilephase. Multivalent cation binding agents such as EDTA fulfill thisrequirement because both the chelator and the multivalent cation bindingagent-metal complex contain charges which make them both water-soluble.Also, neither precipitate when acetonitrile, for example, is added. Thesolubility in aqueous mobile phase can be enhanced by attachingcovalently bound ionic functionality, such as, sulfate, carboxylate, orhydroxy. A preferred multivalent cation binding agent can be easilyremoved from the column by washing with water, organic solvent or mobilephase. Third, the binding agent must not interfere with thechromatographic process.

The multivalent cation binding agent can be a coordination compound.Examples of preferred coordination compounds include water solublechelating agents and crown ethers. Non-limiting examples of multivalentcation binding agents which can be used in the present invention includeacetylacetone, alizarin, aluminon, chloranilic acid, kojic acid, morin,rhodizonic acid, thionalide, thiourea, α-furildioxime, nioxime,salicylaldoxime, dimethylglyoxime, α-furildioxime, cupferron,α-nitroso-β-naphthol, nitroso-R-salt, diphenylthiocarbazone,diphenylcarbazone, eriochrome black T, PAN, SPADNS,glyoxal-bis(2-hydroxyanil), murexide, α-benzoinoxime, mandelic acid,anthranilic acid, ethylenediamine, glycine, triaminotriethylamine,thionalide, triethylenetetramine, EDTA, metalphthalein, arsonic acids,α,α'-bipyridine, 4-hydroxybenzothiazole, 8-hydroxyquinaldine,8-hydroxyquinoline, 1,10-phenanthroline, picolinic acid, quinaldic acid,α,α',α"-terpyridyl, 9-methyl-2,3,7-trihydroxy-6-fluorone, pyrocatechol,salicylic acid, tiron, 4-chloro-1,2-dimercaptobenzene, dithiol,mercaptobenzothiazole, rubeanic acid, oxalic acid, sodiumdiethyldithiocarbarbamate, and zinc dibenzyldithiocarbamate. These andother examples are described by Perrin in Organic Complexing Reagents:Structure, Behavior, and Application to Inorganic Analysis, Robert E.Krieger Publishing Co. (1964). In the present invention, a preferredmultivalent cation binding agent is EDTA.

To achieve high resolution chromatographic separations ofpolynucleotides, it is generally necessary to tightly pack thechromatographic column with the solid phase polymer beads. Any knownmethod of packing the column with a column packing material can be usedin the present invention to obtain adequate high resolution separations.Typically, a slurry of the polymer beads is prepared using a solventhaving a density equal to or less than the density of the polymer beads.The column is then filled with the polymer bead slurry and vibrated oragitated to improve the packing density of the polymer beads in thecolumn. Mechanical vibration or sonication are typically used to improvepacking density.

For example, to pack a 50×4.6 mm I.D. column, 2.0 grams of beads can besuspended in 10 mL of methanol with the aid of sonication. Thesuspension is then packed into the column using 50 mL of methanol at8,000 psi of pressure. This improves the density of the packed bed.

The separation method of the invention is generally applicable to thechromatographic separation of single stranded and double strandedpolynucleotides of DNA and RNA. Samples containing mixtures ofpolynucleotides can result from total synthesis of polynucleotides,cleavage of DNA or RNA with restriction endonucleases or with otherenzymes or chemicals, as well as nucleic acid samples which have beenmultiplied and amplified using polymerase chain reaction techniques.

The method of the present invention can be used to separate doublestranded polynucleotides having up to about 1500 to 2000 base pairs. Inmany cases, the method is used to separate polynucleotides having up to600 bases or base pairs, or which have up to 5 to 80 bases or basepairs.

In a preferred embodiment, the separation is by Matched IonPolynucleotide Chromatography (MIPC). The nonporous beads of theinvention are used as a reverse phase material that will function withcounterion agents and a solvent gradient to effect the DNA separations.In MIPC, the polynucleotides are paired with a counterion and thensubjected to reverse phase chromatography using the nonporous beads ofthe present invention.

There are several types of counterions suitable for use with MIPC. Theseinclude a mono-, di-, or trialkylamine that can be protonated to form apositive counter charge or a quaternary alkyl substituted amine thatalready contains a positive counter charge. The alkyl substitutions maybe uniform (for example, triethylammonium acetate or tetrapropylammoniumacetate) or mixed (for example, propyldiethylammonium acetate). The sizeof the alkyl group may be small (methyl) or large (up to 30 carbons)especially if only one of the substituted alkyl groups is large and theothers are small. For example octyldimethylammonium acetate is asuitable counterion agent. Preferred counterion agents are thosecontaining alkyl groups from the ethyl, propyl or butyl size range.

The purpose of the alkyl group is to impart a nonpolar character to thepolynucleic acid through a matched ion process so that the polynucleicacid can interact with the nonpolar surface of the separation media. Therequirements for the extent of nonpolarity of the counterion-DNA pairdepends on the polarity of the separation media, the solvent conditionsrequired for separation, the particular size and type of fragment beingseparated. For example, if the polarity of the separation media isincreased, then the polarity of the counterion agent may have to changeto match the polarity of the surface and increase interaction of thecounterion-DNA pair. Triethylammonium acetate is preferred althoughquaternary ammonium reagents such as tetrapropyl or tetrabutyl ammoniumsalts can be used when extra nonpolar character is needed or desired. Ingeneral, as the polarity of the alkyl group is increased, size specificseparations, sequence independent separations become more possible.Quaternary counterion reagents are not volatile, making collection offragments more difficult.

In some cases, it may be desired to increase the range of concentrationof organic solvent used to perform the separation. For example,increasing the alkyl length on the counterion agent will increase thenonpolarity of the counterion-DNA pair resulting in the need to eitherincrease the concentration of the mobile phase organic solvent, orincrease the strength of the organic solvent type, e.g. acetonitrile isabout two times more effective than methanol for eluting polynucleicacids. There is a positive correlation between concentration of theorganic solvent required to elute a fragment from the column and thelength of the fragment. However, at high organic solvent concentrations,the polynucleotide could precipitate. To avoid precipitation, a strongorganic solvent or a smaller counterion alkyl group can be used. Thealkyl group on the counterion reagent can also be substituted withhalides, nitro groups, or the like to moderate polarity.

The mobile phase preferably contains a counterion agent. Typicalcounterion agents include trialkylammonium salts of organic or inorganicacids, such as lower alkyl primary, secondary, and lower tertiaryamines, lower trialkyammonium salts and lower quatemary alkyalmmoniumsalts. Lower alkyl refers to an alkyl radical of one to six carbonatoms, as exemplified by methyl, ethyl, n-butyl, i-butyl, t-butyl,isoamyl, n-pentyl, and isopentyl. Examples of counterion agents includeoctylammonium acetate, octadimethylammonium acetate, decylammoniumacetate, octadecylammonium acetate, pyridiniumammonium acetate,cyclohexylammonium acetate, diethylammonium acetate, propylethylammoniumacetate, propyldiethylammonium acetate, butylethylammonium acetate,methylhexylammonium acetate, tetramethylammonium acetate,tetraethylammonium acetate, tetrapropylammonium acetate,tetrabutylammonium acetate, dimethydiethylammonium acetate,triethylammonium acetate, tripropylammonium acetate, tributylammoniumacetate, tetrapropylammonium acetate, and tetrabutylammonium acetate.Although the anion in the above examples is acetate, other anions mayalso be used, including carbonate, phosphate, sulfate, nitrate,propionate, formate, chloride, and bromide, or any combination of cationand anion. These and other agents are described by Gjerde, et al. in IonChromatography, 2nd Ed., Dr. Alfred Huthig Verlag. Heidelberg (1987).Counterion agents that are volatile are preferred for use in the methodof the invention, with triethylammonium acetate (TEAA) andtriethylammonium hexafluoroisopropyl alcohol being most preferred.

To achieve optimum peak resolution during the separation of DNA by MIPCusing the beads of the invention, the method is preferably performed ata temperature within the range of 20° C. to 90° C.; more preferably, 30°C. to 80° C.; most preferably, 50° C. to 75° C. The flow rate isselected to yield a back pressure not exceeding 5000 psi. In general,separation of single-stranded fragments should be performed at highertemperatures.

Applicants have found that the temperature at which the separation isperformed affects the choice of organic solvents used in the separation.One reason is that the solvents affect the temperature at which a doublestranded DNA will melt to form two single strands or a partially meltedcomplex of single and double stranded DNA. Some solvents can stabilizethe melted structure better than other solvents. The other reason asolvent is important is because it affects the distribution of the DNAbetween the mobile phase and the stationary phase. Acetonitrile and1-propanol are preferred solvents in these cases. Finally, the toxicity(and cost) of the solvent can be important. In this case, methanol ispreferred over acetonitrile and 1-propanol is preferred over methanol.

When the separation is performed at a temperature within the aboverange, an organic solvent that is water soluble is preferably used, forexample, alcohols, nitriles, dimethylformamide (DMF), tetrahydrofuran(THF), esters, and ethers. Water soluble solvents are defined as thosewhich exist as a single phase with aqueous systems under all conditionsof operation of the present invention. Solvents which are particularlypreferred for use in the method of this invention include methanol,ethanol, 2-propanol, 1-propanol, tetrahydrofuran (THF), andacetonitrile, with acetonitrile being most preferred overall.

Mixtures of polynucleotides in general, and double stranded DNA inparticular, are effectively separated using Matched Ion PolynucleotideChromatography (MIPC). MIPC separations of polynucleotides atnon-denaturing temperature, typically less than about 50° C., are basedon base pair length. However, even traces of multivalent cationsanywhere in the solvent flow path can cause a significant deteriorationin the resolution of the separation after multiple uses of an MIPCcolumn. This can result in increased cost caused by the need to purchasereplacement columns and increased downtime.

Therefore, effective measures are preferably taken to preventmultivalent metal cation contamination of the separation systemcomponents, including separation media and mobile phase contacting.These measures include, but are not limited to, washing protocols toremove traces of multivalent cations from the separation media andinstallation of guard cartridges containing cation capture resins, inline between the mobile phase reservoir and the MIPC column. These, andsimilar measures, taken to prevent system contamination with multivalentcations have resulted in extended column life and reduced analysisdowntime.

Recently, MIPC has been successfully applied to the detection ofmutations in double stranded DNA by separating heteroduplexes fromhomoduplexes as described in co-pending U.S. patent application Ser. No.09/129,105 filed Aug. 4, 1998 which is herein incorporated by reference.Such separations depend on the lower temperature required to denature aheteroduplex at the site of base pair mismatch compared to a fullycomplimentary homoduplex DNA fragment. MIPC, when performed at atemperature which is sufficient to partially denature a heteroduplex isreferred to herein as Denaturing Matched Ion PolynucleotideChromatography (DMIPC). DMIPC is typically performed at a temperaturebetween 52° C. and 70° C. The optimum temperature for performing DMIPCis 54° C. to 59° C.

The previously described precautions taken to remove multivalent metalcations were adequate for maintaining column life, as demonstrated bygood separation efficiency, under non-denaturing conditions. However,Applicants have surprisingly found that when performed at partiallydenaturing temperature, conditions for effective DMIPC separationsbecome more stringent. For example, a separation of a standard pUC18HaeIII digest on a MIPC column at 50° C. provided a good separation ofall the DNA fragments in the digest. However, a standard 209 bp DYS271mutation detection mixture of homoduplexes and heteroduplexes, preparedas described in Example 15, applied to the same MIPC column and elutedunder DMIPC conditions, i.e., 56° C., afforded a poor separation themixture components. In order to optimize column life and maintaineffective separation performance of homoduplexes from heteroduplexes atpartially denaturing temperatures, as is required for mutationdetection, special column washing and storage procedures are used in theembodiments of the invention as described hereinbelow.

In one aspect of this invention, therefore, an aqueous solution ofmultivalent cation binding agent is flowed through the column tomaintain separation efficiency. In order to maintain the separationefficiency of a MIPC column, the column is preferably washed withmultivalent cation binding agent solution after about 500 uses or whenthe performance starts to degrade. Examples of suitable cation bindingagents are as described hereinabove.

The concentration of a solution of the cation binding agent can bebetween 0.01 M and 1 M. In a preferred embodiment, the column washingsolution contains EDTA at a concentration of about 0.03 to 0.1 M.

In another embodiment, the solution contains an organic solvent selectedfrom the group consisting of acetonitrile, ethanol, methanol,2-propanol, and ethyl acetate. A preferred solution contains at least 2%organic solvent to prevent microbial growth. In a most preferredembodiment a solution containing 25% acetonitrile is used to wash a MIPCcolumn. The multivalent cation binding solution can contain a counterionagent as described hereinabove.

In one embodiment of a column washing procedure, the MIPC separationcolumn is washed with the multivalent cation binding solution at anelevated temperature in the range of 50° C. to 80° C. In a preferredembodiment the column is washed with a solution containing EDTA, TEAA,and acetonitrile, in the 70° C. to 80° C. temperature range. In aspecific embodiment, the solution contains 0.032 M EDTA, 0.1 M TEAA, and25% acetonitrile.

Column washing can range from 30 seconds to one hour. For example, in ahigh throughput DMIPC assay, the column can be washed for 30 secondsafter each sample, followed by equilibration with mobile phase. SinceDMIPC can be automated by computer, the column washing procedure can beincorporated into the mobile phase selection program without additionaloperator involvement. In a preferred procedure, the column is washedwith multivalent cation binding agent for 30 to 60 minutes at a flowrate preferably in the range of about 0.05 to 1.0 mL/min.

In one embodiment, a DMIPC column is tested with a standard mutationdetection mixture of homoduplexes and heteroduplexes after about 1000sample analyses. If the separation of the standard mixture hasdeteriorated compared to a freshly washed column, then the column can bewashed for 30 to 60 minutes with the multivalent cation binding solutionat a temperature above about 50° C. to restore separation performance.

Applicants have found that other treatments for washing a column canalso be used alone or in combination with those indicated hereinabove.These include: use of high pH washing solutions (e.g., pH 10-12), use ofdenaturants such as urea or formamide, and reverse flushing the columnwith washing solution.

In another aspect, Applicants have discovered that column separationefficiency can be preserved by storing the column separation media inthe column containing a solution of multivalent cation binding agenttherein. The solution of binding agent may also contain a counterionagent. Any of the multivalent cation binding agents, counterion agents,and solvents described hereinabove are suitable for the purpose ofstoring a MIPC column. In a preferred embodiment, a column packed withMIPC separation media is stored in an organic solvent containing amultivalent cation binding agent and a counterion agent. An example ofthis preferred embodiment is 0.032 M EDTA and 0.1M TEAA in 25% aqueousacetonitrile. In preparation for storage, a solution of multivalentcation binding agent, as described above, is passed through the columnfor about 30 minutes. The column is then disconnected from the HPLCapparatus and the column ends are capped with commercially availablethreaded end caps made of material which does not release multivalentcations. Such end caps can be made of coated stainless steel, titanium,organic polymer or any combination thereof.

The effectiveness of the surprising discovery made by Applicants, thatwashing a MIPC column with a multivalent cation binding agent restoresthe ability of the column to separate heteroduplexes and homoduplexes inmutation detection protocols under DMIPC conditions, is described inExample 14 and demonstrated in FIGS. 18, 19, and 20. As described inExample 14, Applicants noticed a decrease in resolution of homoduplexesand heteroduplexes during the use of a MIPC column in mutationdetection. However, no apparent degradation in resolution was observedwhen a DNA standard containing pUC18 HaeIII digest (Sigma/AldrichChemical Co.) was applied at 50° C. (not shown). In order to furthertest the column performance, a mixture of homoduplexes andheteroduplexes in a 209 bp DNA standard was applied to the column underDMIPC conditions of 56° C. (Kuklin et al., Genetic Testing 1:201 (1998).It was surprisingly observed the peaks representing the homoduplexes andheteroduplexes of the mutation detection standard were poorly resolved(FIG. 18).

FIG. 19 shows some improvement in the separation of homoduplexes andheteroduplexes of the standard mutation detection mixture when a guardcartridge containing cation capture resin was deployed in line betweenthe solvent reservoir and the MIPC system. The chromatography shown inFIG. 19 was performed at 56° C. The column used in FIG. 19 was the samecolumn used in the separation shown in FIG. 18 and for separating thestandard pUC18 HaeIII digest.

FIG. 20 shows the separation of homoduplexes and heteroduplexes of thestandard mutation detection mixture at 56° C. on the same column used togenerate the chromatograms in FIGS. 18 and 19. However, in FIG. 20 thecolumn was washed for 45 minutes with a solution comprising 32 mM EDTAand 0.1M TEAA in 25% acetonitrile at 75° C. priorto sample application.FIG. 20 shows four cleanly resolved peaks representing the twohomoduplexes and the two heteroduplexes of the standard 209 bp mutationdetection mixture. This restoration of the separation ability, afterwashing with a solution containing a cation binding agent, of the MIPCcolumn under DMIPC conditions compared to the chromatograms of FIGS. 18and 19 clearly shows the effectiveness and the utility of the presentinvention.

In an important aspect of the present invention, Applicants havedeveloped a standardized criteria to evaluate the performance of a DMIPCseparation media. DMIPC as used herein, is defined as a process forseparating heteroduplexes and homoduplexes using a non-polar separationmedium (e.g., beads or rod) in the column, wherein the process uses acounterion agent, and an organic solvent to desorb the nucleic acid fromthe medium, and wherein the medium is characterized as having a MutationSeparation Factor (MSF) of at least 0.1. In one embodiment, the mediumhas a Mutation Separation Factor of at least 0.2. In a preferredembodiment, the medium has a Mutation Separation Factor of at least 0.5.In an optimal embodiment, the medium has a Mutation Separation Factor ofat least 1.0.

The performance of the column is demonstrated by high efficiencyseparation by DMIPC of heteroduplexes and homoduplexes. Applicants havefound that the best criterion for measuring performance is a MutationSeparation Factor as described in Example 13. This is measured as thedifference between the areas of the resolved heteroduplex and homoduplexpeaks. A correction factor may be applied to the generated areasunderneath the peaks. The following aspects may affect the calculatedareas of the peaks and reproducibility of the same: baseline drawn, peaknormalization, inconsistent temperature control, inconsistent elutionconditions, detector instability, flow rate instability, inconsistentPCR conditions, and standard and sample degradation. Some of theseaspects are discussed by Snyder, et al., in Introduction to ModernLiquid Chromatography, 2^(nd) Ed., John Wiley and Sons, pp. 542-574(1979) which is incorporated by reference herein.

The Mutation Separation Factor (MSF) is determined by the followingequation:

    MSF=(area peak 2-area peak 1)/area peak 1

where area peak 1 is the area of the peak measured after DMIPC analysisof wild type and area peak 2 is the total area of the peak or peaksmeasured after DMIPC analysis of a hybridized mixture containing aputative mutation, with the hereinabove correction factors taken intoconsideration, and where the peak heights have been normalized to thewild type peak height. Separation particles are packed in an HPLC columnand tested for their ability to separate a standard hybridized mixturecontaining a wild type 100 bp Lambda DNA fragment and the corresponding100 bp fragment containing an A to C mutation at position 51.

High pressure pumps are used for pumping mobile phase in the systemsdescribed in U.S. Pat. No. 5,585,236 to Bonn and in U.S. Pat. No.5,772,889 to Gjerde. It will be appreciated that other methods are knownfor driving mobile phase through separation media and can be used incarrying out the separations of polynucleotides as described in thepresent invention. A non-limiting example of such an alternative methodincludes "capillary electrochromatography" (CEC) in which an electricfield is applied across capillary columns packed with microparticles andthe resulting electroosmotic flow acts as a pump for chromatography.Electroosmosis is the flow of liquid, in contact with a solid surface,under the influence of a tangentially applied electric field. Thetechnique combines the advantages of the high efficiency obtained withcapillary electrophoretic separations, such as capillary zoneelectrophoresis, and the general applicability of HPLC. CEC has thecapability to drive the mobile phase through columns packed withchromatographic particles, especially small particles, when usingelectroosmotic flow. High efficiencies can be obtained as a result ofthe plug-like flow profile. In the use of CEC in the present invention,solvent gradients are used and rapid separations can be obtained usinghigh electric fields. The following references describing CEC are eachincorporated in their entirety herein: Dadoo, et al, LC-GC 15:630(1997); Jorgenson, et al., J. Chromatog. 218:209 (1981); Pretorius, etal., J. Chromatog. 99:23 (1974); and the following U.S. Pat. Nos. toDadoo 5,378,334 (1995), U.S. Pat. No. 5,342,492 (1994), and U.S. Pat.No. 5,310,463 (1994). In the operation of this aspect of the presentinvention, the capillaries are packed, either electrokinetically orusing a pump, with the separation beads described in the presentspecification. In another embodiment, a polymeric rod is prepared bybulk free radical polymerization within the confines of a capillarycolumn. Capillaries are preferably formed from fused silica tubing oretched into a block. The packed capillary (e.g., a 150-μm i.d. with a20-cm packed length and a window located immediately before the outletfrit) is fitted with frits at the inlet and outlet ends. An electricfield, e.g., 2800V/cm, is applied. Detection can be by uv absorbance orby fluorescence. A gradient of organic solvent, e.g., acetonitrile, isapplied in a mobile phase containing counterion agent (e.g. 0.1 M TEAA).to elute the polynucleotides. The column temperature is maintained byconventional temperature control means. In the preferred embodiment, allof the precautions for minimizing trace metal contaminants as describedhereinabove are employed in using CEC.

In a related method, mixtures of polynucleotides are separated on thinlayer chromatography (TLC) plates. In this method, the beads of thepresent invention are mixed with a binder and bound to a TLC plate byconventional methods (Remington: The Science and Practice of Pharmacy,19^(th) Edition, Gennaro ed., Mack Publishing Co. (1995) pp. 552-554). Afluorophore is optionally included in the mixture to facilitatedetection. The sample is spotted on the plate and the sample is runisocratically under capillary flow. In a preferred embodiment, thesample is run under electroosmotic flow in a process called High-SpeedTLC (HSTLC). In the case of HSTLC, the plate is first wetted withsolvent (e.g., acetonitrile solution in the presence of counterionagent) and an electric field (e.g., 2000 V/cm) is applied. Solventaccumulating at the top of the plate is removed by suction. Applicantshave surprisingly discovered that dDNA of selected ranges of base pairlength are separable under isocratic conditions by MIPC using the beadsof the present invention as described in Example 6. The isocraticsolvent conditions for separating a selected range of DNA base pairlength, as determined using MIPC, are used in the TLC and HSTLC methods.

Applicants have determined that the chromatographic separations ofdouble stranded DNA fragments exhibit unique Sorption Enthalpies(ΔH_(sorp)). Two compounds (in this case, DNA fragments of differentsize) can only be separated if they have different partitioncoefficients (K). The Nernst partition coefficient is defined as theconcentration of an analyte (A) in the stationary phase divided by itsconcentration in the mobile phase: ##EQU1## The partition coefficient(K) and the retention factor (k) are related through the followingequations: ##EQU2## the quotient V_(m) /V_(s) is also called phasevolume ratio (Φ). Therefore:

    k=KΦ

To calculate the sorption enthalpies, the following fundamentalthermodynamic equations are necessary: ##EQU3## By transforming the lasttwo equations, one obtains the Van't Hoff equation: ##EQU4## From a plotIn k versus 1/T, the sorption enthalpy ΔH_(sorp) can be obtained fromthe slope of the graph (if a straight line is obtained). ΔS_(sorp) canbe calculated if the phase volume ratio (Φ) is known.

The Sorption Enthalpy ΔH_(sorp) is positive (ΔH_(sorp) >0) showing theseparation is endothermic using acetonitrile as the solvent (FIGS. 3 and4), and using methanol as the solvent, the Sorption Enthalpy ΔH_(sorp)is negative (ΔH_(sorp) <0), showing the separation is exothermic (FIG.5).

The thermodynamic data (as shown in the Examples hereinbelow) reflectthe relative affinity of the DNA-counterion agent complex for the beadsof the invention and the elution solvent. An endothermic plot indicatesa preference of the DNA complex for the bead. An exothermic plotindicates a preference of the DNA complex for the solvent over the bead.The plots shown herein are for alkylated and non-alkylated surfaces asdescribed in the Examples. Most liquid chromatographic separations showexothermic plots.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not intended to be limitingthereof.

Procedures described in the past tense in the examples below have beencarried out in the laboratory. Procedures described in the present tensehave not yet been carried out in the laboratory, and are constructivelyreduced to practice with the filing of this application.

EXAMPLE 1 Preparation of nonporous poly(styrene-divinylbenzene)particles

Sodium chloride (0.236 g) was added to 354 mL of deionized water in areactor having a volume of 1.0 liter. The reactor was equipped with amechanical stirrer, reflux condenser, and a gas introduction tube. Thedissolution of the sodium chloride was carried out under inertatmosphere (argon), assisted by stirring (350 rpm), and at an elevatedtemperature (87° C.). Freshly distilled styrene (33.7 g) and 0.2184 g ofpotassium peroxodisulfate (K₂ S₂ O₈) dissolved in 50 mL of deionizedwater were then added. Immediately after these additions, the gasintroduction tube was pulled out of the solution and positioned abovethe liquid surface. The reaction mixture was subsequently stirred for6.5 hours at 87° C. After this, the contents of the reactor were cooleddown to ambient temperature and diluted to a volume yielding aconcentration of 54.6 g of polymerized styrene in 1000 mL volume ofsuspension resulting from the first step. The amount of polymerizedstyrene in 1000 mL was calculated to include the quantity of the polymerstill sticking to the mechanical stirrer (approximately 5-10 g). Thediameter of the spherical beads in the suspension was determined bylight microscopy to be about 1.0 micron.

Beads resulting from the first step are still generally too small andtoo soft (low pressure stability) for use as chromatographic packings.The softness of these beads is caused by an insufficient degree ofcrosslinking. In a second step, the beads are enlarged and the degree ofcrosslinking is increased.

The protocol for the second step is based on the activated swellingmethod described by Ugelstad et al. (Adv. Colloid Interface Sci.,13:101-140 (1980)). In order to initiate activated swelling, or thesecond synthetic step, the aqueous suspension of polystyrene seeds (200ml) from the first step was mixed first with 60 mL of acetone and thenwith 60 mL of a 1-chlorododecane emulsion. To prepare the emulsion,0.206 g of sodium dodecylsulfate, 49.5 mL of deionized water, and 10.5mL of 1-chlorododecane were brought together and the resulting mixturewas kept at 0° C. for 4 hours and mixed by sonication during the entiretime period until a fine emulsion of <0.3 microns was obtained. Themixture of polystyrene seeds, acetone, and 1-chlorododecane emulsion wasstirred for about 12 hours at room temperature, during which time theswelling of the beads occurred. Subsequently, the acetone was removed bya 30 minute distillation at 80° C.

Following the removal of acetone, the swollen beads were further grownby the addition of 310 g of a ethyidivinylbenzene and divinylbenzene(DVB) (1:1.71) mixture also containing 2.5 g of dibenzoylperoxide as aninitiator. The growing occurred with stirring and with occasionalparticle size measurements by means of light microscopy.

After completion of the swelling and growing stages, the reactionmixture was transferred into a separation funnel. In an unstirredsolution, the excess amount of the monomer separated from the layercontaining the suspension of the polymeric beads and could thus beeasily removed. The remaining suspension of beads was returned to thereactor and subjected to a stepwise increase in temperature (63° C. forabout 7 hours, 73° C. for about 2 hours, and 83° C. for about 12 hours),leading to further increases in the degree of polymerization (>500). Thepore size of beads prepared in this manner was below the detection limitof mercury porosimetry (<30 Å).

After drying, the dried beads (10 g) from step two were washed fourtimes with 100 mL of n-heptane, and then two times with each of thefollowing: 100 mL of diethylether, 100 mL of dioxane, and 100 mL ofmethanol. Finally, the beads were dried.

EXAMPLE 2 Acid Wash Treatment

The beads prepared in Example 1 were washed three times withtetrahydrofuran and two times with methanol. Finally the beads werestirred in a mixture containing 100 mL tetrahydrofuran and 100 mLconcentrated hydrochloric acid for 12 hours. After this acid treatment,the polymer beads were washed with a tetrahydrofuran/water mixture untilneutral (pH=7). The beads were then dried at 40° C. for 12 hours.

EXAMPLE 3 Standard Procedure for Testing the Performance of SeparationMedia

Separation particles are packed in an HPLC column and tested for theirability to separate a standard DNA mixture. The standard mixture is apUC18 DNA-HaeIII digest (Sigma-Aldrich, D6293) which contains 11fragments having 11, 18, 80, 102, 174, 257, 267, 298, 434, 458, and 587base pairs, respectively. The standard is diluted with water and fiveμL, containing a total mass of DNA of 0.25 μg, is injected.

Depending on the packing volume and packing polarity, the procedurerequires selection of the driving solvent concentration, pH, andtemperature. The separation conditions are adjusted so that theretention time of the 257, 267 peaks is about 6 to 10 minutes. Any oneof the following solvents can be used: methanol, ethanol, 2-propanol,1-propanol, tetrahydrofuran (THF), or acetonitrile. A counterion agentis selected from trialkylamine acetate, trialkylamine carbonate,trialkylamine phosphate, or any other type of cation that can form amatched ion with the polynucleotide anion.

As an example of this procedure, FIG. 2 shows the high resolution of thestandard DNA mixture using octadecyl modified, nonporouspoly(ethylvinylbenzene-divinylbenzene) beads. The separation wasconducted under the following conditions: Eluent A: 0.1 M TEAA, pH 7.0;Eluent B: 0.1 M TEAA, 25% acetonitrile; Gradient:

    ______________________________________                                        Time (min)       % A    % B                                                   ______________________________________                                         0.0             65     35                                                     3.0             45     55                                                    10.0             35     65                                                    13.0             35     65                                                    14.0              0     100                                                   15.5              0     100                                                   16.5             65     35                                                    ______________________________________                                    

The flow rate was 0.75 mL/min, detection UV at 260 nm, column temp. 50°C. The pH was 7.0.

As another example of this procedure using the same separationconditions as in FIG. 2, FIG. 3 is a high resolution separation of thestandard DNA mixture on a column containing nonporous 2.1 micron beadsof underivatized poly(styrene-divinylbenzene).

EXAMPLE 4 Sorption Enthalpy Measurements

Four fragments (174 bp, 257 bp, 267 bp, and 298 bp, found in 5 μL pUC18DNA-HaeIII digest, 0.04 μg DNA/μL) were separated under isocraticconditions at different temperatures using octadecyl modified, nonporouspoly(styrene-divinylbenzene) polymer beads. The separation was carriedout using a Transgenomic WAVE™ DNA Fragment Analysis System equippedwith a DNASep™ column (Transgenomic, Inc., San Jose, Calif.) under thefollowing conditions: Mobile phase: 0.1 M triethylammonium acetate,14.25% (v/v) acetonitrile at 0.75 mL/min, detection at 250 nm UV,temperatures at 35, 40, 45, 50, 55, and 60° C., respectively. A plot ofIn k versus 1/T shows that the retention factor k is increasing withincreasing temperature (FIG. 4). This indicates that the retentionmechanism is based on an endothermic process (ΔH_(sorp) >0).

The same experiments on non-alkylated poly(styrene-divinylbenzene) beadsgave a negative slope for a plot of In k versus 1/T, although the plotis slightly curved (FIG. 5).

The same experiments performed on octadecyl modified, nonporouspoly(styrene-divinylbenzene) beads but with methanol replacing theacetonirile as solvent gave a plot In k versus 1/T showing the retentionfactor k is decreasing with increasing temperature (FIG. 6). Thisindicates the retention mechanism is based on an exothermic process(ΔH_(sorp) <0).

EXAMPLE 5 Separations with alkylatedpoly(styrene-divinylbenzene) beads

Mobile phase components are chosen to match the desorption ability ofthe elution solvent in the mobile phase to the attraction properties ofthe bead to the DNA-counterion complex. As the polarity of the beaddecreases, a stronger (more organic) or higher concentration of solventwill be required. Weaker organic solvents such as methanol are generallyrequired at higher concentrations than stronger organic solvents such asacetonitrile.

FIG. 7 shows the high resolution separation of DNA restriction fragmentsusing octadecyl modified, nonporouspoly(ethylvinylbenzene-divinylbenzene) beads. The experiment wasconducted under the following conditions: Column: 50×4.6 mm I.D.; mobilephase 0.1 M TEAA, pH 7.2; gradient: 33-55% acetonitrile in 3 min, 55-66%acetonitrile in 7 min, 65% acetonitrile for 2.5 min; 65-100%acetonitrile in 1 min; and 100-35% acetonitrile in 1.5 min. The flowrate was 0.75 mL/min, detection UV at 260 nm, column temp. 51° C. Thesample was 5 μL (=0.2 μg pUC18 DNA-HaeIII digest).

Repeating the procedure of FIG. 7 replacing the acetonitrile with 50.0%methanol in 0.1 M (TEAA) gave the separation shown in FIG. 8.

Repeating the procedure of FIG. 7 replacing the acetonitrile with 25.0%ethanol in 0.1 M (TEM) gave the separation shown in FIG. 9.

Repeating the procedure of FIG. 7 replacing the acetonitrile with 25%vodka (Stolichnaya, 100 proof) in 0.1 M (TEAA) gave the separation shownin FIG. 10.

The separation shown in FIG. 11 was obtained using octadecyl modified,nonporous poly(ethylvinylbenzene-divinylbenzene) beads as follows:Column: 50×4.6 mm I.D.; mobile phase 0.1 M tetraethylacetic acid (TEAA),pH 7.3; gradient: 12-18% 0.1 M TEAA and 25.0% 1-propanol (Eluent B) in 3min, 18-22% B in 7 min, 22% B for 2.5 min; 22-100% B in 1 min; and100-12% B in 1.5 min. The flow rate was 0.75 mL/min, detection UV at 260nm, and column temp. 51° C. The sample was 5 μL (=0.2 μg pUC18DNA-HaeIII digest).

The separation shown in FIG. 12 was obtained using octadecyl modified,nonporous poly(ethylvinylbenzene-divinylbenzene) beads as follows:Column: 50×4.6 mm I.D.; mobile phase 0.1 M TEAA, pH 7.3; gradient:15-18% 0.1 M TEAA and 25.0% 1-propanol (Eluent B) in 2 min, 18-21% B in8 min, 21% B for 2.5 min; 21-100% B in 1 min; and 100-15% B in 1.5 min.The flow rate was 0.75 mL/min, detection UV at 260 nm, and column temp.51° C. The sample was 5 μL (=0.2 μg pUC18 DNA-HaeIII digest).

The separation shown in FIG. 13 was obtained using octadecyl modified,nonporous poly(ethylvinylbenzene-divinylbenzene) beads as follows:Column: 50×4.6 mm I.D.; mobile phase 0.1 M TEAA, pH 7.3; gradient:35-55% 0.1 M TEAA and 10.0% 2-propanol (Eluent B) in 3 min, 55-65 % B in10 min, 65% B for 2.5 min; 65-100% B in 1 min; and 100-35% B in 1.5 min.The flow rate was 0.75 mL/min, detection UV at 260 nm, and column temp.51° C. The sample was 5 μL (=0.2 μg pUC18 DNA-HaeIII digest).

The separation shown in FIG. 14 was obtained using octadecyl modified,nonporous poly(ethylvinylbenzene-divinylbenzene) beads as follows:Column: 50×4.6 mm I.D.; mobile phase 0.1 M TEA₂ HPO₄, pH 7.3; gradient:35-55% 0.1 M TEA₂ HPO₄ and 10.0% 2-propanol (Eluent B) in 3 min, 55-65%B in 7 min, 65% B for 2.5 min; 65-100% B in 1 min; and 100-65% B in 1.5min. The flow rate was 0.75 mL/min, detection UV at 260 nm, and columntemp. 51° C. The sample was 5 μL (=0.2 μg pUC18 DNA-HaeIII digest).

The separation shown in FIG. 15 was obtained using octadecyl modified,nonporous poly(ethylvinylbenzene-divinylbenzene) beads as follows:Column: 50×4.6 mm I.D.; mobile phase 0.1 M TEAA, pH 7.3; gradient: 6-9%0.1 M TEAA and 25.0% THF (Eluent B) in 3 min, 9-11% B in 7 min, 11% Bfor 2.5 min; 11-100% B in 1 min; and 100-6% B in 1.5 min. The flow ratewas 0.75 mL/min, detection UV at 260 nm, and column temp. 51 ° C. Thesample was 5 μL (=0.2 μg pUC18 DNA-HaeIII digest).

EXAMPLE 6 Isocratic/gradient separation of ds DNA

The following is an isocratic/gradient separation of ds DNA usingnonporous poly(styrene-divinylbenzene) beads. Isocratic separations havenot been performed in DNA separations because of the large differencesin the selectivity of DNA/alkylammonium ion pair for beads. However, byusing a combination of gradient and isocratic elution conditions, theresolving power of a system can be enhanced for a particular size rangeof DNA. For example, the range of 250-300 base pairs can be targeted byusing a mobile phase of 0.1 M TEAA, and 14.25% acetonitrile at 0.75mL/min at 40° C. on 50×4.6 mm cross-linked poly(styrene-divinylbenzene)column, 2.1 micron. 5 μL of pUC18 DNA-HaeIII digest (0.2 μg) wasinjected under isocratic conditions and 257, 267 and 298 base pairs DNAeluted completely resolved as shown in FIG. 16. Then the column wascleaned from larger fragments with 0.1 M TEAA/25% acetonitrile at 9minutes. In other examples, there might be an initial isocratic step (tocondition the column), then a gradient step (to remove or target thefirst group of DNA at a particular size), then an isocratic step (toseparate the target material of a different size range) and finally agradient step to clean the column.

EXAMPLE 7 Bromination of Remaining Double Bonds on the Surface ofPoly(Styrene-Divinylbenzene) Polymer Beads

50.0 g of a poly(styrene-divinylbenzene) polymer beads were suspended in500 g of tetrachloromethane. The suspension was transferred into a 1000mL glass reactor (with attached reflux condenser, separation funnel andoverhead stirrer). The mixture was kept at 20° C. Bromine (100 mL) wasadded over a period of 20 minutes. After addition was completed,stirring continued for 60 minutes. The temperature was raised to 50° C.to complete the reaction (2 hours).

The polymer beads were separated from the tetrachloromethane and excessbromine by means of centrifugation and cleaned with tetrahydrofuran(once with 100 mL) and methanol (twice with 100 mL). The polymer beadswere dried at 40° C.

The polymer beads are packed into a 50×4.6 mm i.d column and the DNASeparation Factor is greater than 0.05 as tested by the procedure ofExample 3.

EXAMPLE 8 Nitration of a Poly(Styrene-Divinylbenzene) Polymer Beads

In a 1000 mL glass reactor 150 mL of concentrated nitric acid (65%) werecombined with 100 mL concentrated sulfuric acid. The acid mixture wascooled to 0-4° C. When the temperature had dropped to <4° C., 50 g ofpoly(styrene-divinylbenzene) polymer beads were added slowly undercontinuous stirring. After addition was completed, 50 mL of nitric acid(65%) was added. The suspension was stirred for three hours, maintaininga temperature of 5-10° C.

On the next day the reaction was quenched by adding ice to thesuspension. The polymer beads were separated from the acid by means ofcentrifugation. The polymer beads were washed to neutrality with water,followed by washing steps with tetrahydrofurane (four times with 100 mL)and methanol (four times with 100 mL). The polymer beads were dried at40° C.

The polymer beads are packed into a 50×4.6 mm i.d column and the DNASeparation Factor is greater than 0.05 as tested by the procedure ofExample 3.

EXAMPLE 9 Preparation of a Non-Polar Organic Polymer MonolithChromatography Column

A chromatography tube in which the monolith polymeric separation mediumis prepared is made of stainless steel. The monomers, styrene(Sigma--Aldrich Chemical Corp.) and divinylbenzene (Dow Chemical Corp.)are dried over magnesium sulfate and distilled under vacuum.

To a solution of a 1:1 mixture by volume of the distilled styrene anddivinylbenzene, containing 1% by weight (with respect to monomers) ofazobisisobutyronitrile (AIBN), is added eight volumes of a solution ofthe porogenic solvent, dodecyl alcohol and toluene (70:30). The solutionso prepared is bubbled with nitrogen for 15 minutes and is used to filla chromatography tube (50×8 mm I.D.) sealed with a rubber nut plug atthe bottom end. The tube is then sealed at the top end with a rubber nutplug and the contents are allowed to polymerize at 70° C. for 24 hours.

Following polymerization, the rubber plugs are replaced by column endfittings and the column is connected to an HPLC system. The HPLCinstrument has a low-pressure mixing quaternary gradient capability. Acartridge or guard column containing an iminodiacetate multivalentcation capture resin is placed in line between the column and the mobilephase source reservoir. The column is then washed by flowing 100 mL oftetrahydrofuran (THF) at 1 mL/min through the column to remove thedodecyl alcohol and toluene, thereby creating through-pores in theotherwise solid polymer monolith.

In this example, all of the flow paths are either titanium, sapphire,ceramic, or PEEK, except for the tube body, which is 316 stainlesssteel. The interior of the 316 stainless steel tube is passivated withdilute nitric acid prior to use.

EXAMPLE 10 Acid Wash Treatment To Remove Multivalent Metal CationContaminants

The non-polar, organic polymer monolith column is washed by flowingtetrahydrofuran through the column at a flow rate of 2 mL per minute for10 minutes followed by flowing methanol through the column at 2 mL perminute for 10 minutes. The non-polar, organic polymer monolith column iswashed further by flowing a mixture containing 100 mL of tetrahydrofuranand 100 mL of concentrated hydrochloric acid through the column at 10 mLper minute for 20 minutes. Following this acid treatment, the non-polar,organic polymer monolith column is washed by flowingtetrahydrofuran/water (1:1) through the column at 2 mL per minute untilneutral (pH 7).

EXAMPLE 11 Bromination of Remaining Double Bonds on the Surface ofNon-Polar Organic Polymer Monolith Column

Any double bonds remaining on the surface of the monolith columnprepared in Example 9 are reacted with bromine as described in Example7.

EXAMPLE 12 Nitration of a Non-Polar Organic Polymer Monolith Column

The non-polar organic polymer column prepared in Example 9 is nitratedas described in Example 8.

EXAMPLE 13 Determination of the Mutation Separation Factor

The Mutation Separation Factor (MSF) is determined by the followingequation:

    MSF=(area peak 2-area peak 1)/area peak 1

where area peak 1 is the area of the peak measured after DMIPC analysisof wild type and area peak 2 is the total area of the peak or peaksmeasured after DMIPC analysis of a hybridized mixture containing aputative mutation, with the hereinabove correction factors taken intoconsideration, and where the peak heights have been normalized to thewild type peak height. Separation particles are packed in an HPLC columnand tested for their ability to separate a standard hybridized mixturecontaining a wild type 100 bp Lambda DNA fragment and the corresponding100 bp fragment containing an A to C mutation at position 51.

Depending on the packing volume and packing polarity, the procedurerequires selection of the driving solvent concentration, pH, andtemperature. Any one of the following solvents can be used:acetonitrile, tetrahydrofuran, methanol, ethanol, or propanol. Any oneof the following counterion agents can be used: trialkylamine acetate,trialkylamine carbonate, and trialkylamine phosphate.

As an example of the determination of the Mutation Separation Factor,FIG. 22 shows the resolution of the separation of the hybridized DNAmixture.

The PCR conditions used with each of the primers are described in thetable below. All the components were combined and vortexed to ensuregood mixing, and centrifuged. Aliquots were then distributed into PCRtubes as shown in the following table:

    ______________________________________                                        COMPONENT               VOLUME                                                ______________________________________                                        Pfu 10X Buffer (Cat. No.                                                                              5      μL                                          600153-82, Stratagene, Inc.,                                                  La Jolla, CA)                                                                 100 μM dNTP Mix      4      μL                                          Primer 1                7.5    μL                                          (forward)                                                                     Primer 2                8.5    μL                                          (reverse)                                                                     H.sub.2 O               19.5   μL                                          Lambda DNA Template     5      μL                                          PFUTurbo ™           0.5    μL                                          (600250, Stratagene)                                                          ______________________________________                                    

The PCR tubes were placed into a thermocycler (PTC-100 PrograrmmableThermal Controller from MJ Research, Inc., Watertown, Mass.) and thetemperature cycling program was initiated. The cycling programparameters are shown in the table below:

    ______________________________________                                        STEP        TEMPERATURE    TIME                                               ______________________________________                                        1           94° C.  2 minutes                                          2           94° C.  1 minute                                           3           58° C.  1 minute                                           4           72° C.  1 minute                                           5           Go to Step 2, 34X                                                 6           72° C.  10 minutes                                         7           End                                                               ______________________________________                                    

The DIMPC conditions used for the mutation detection separations areshown below:

Eluent A: 0.1 M TEAA; Eluent B: 0.1 M TEAA, 25% Acetonitrile; Flow rate:0.90 mL/min; Gradient:

    ______________________________________                                        Time (min)       % A    % B                                                   ______________________________________                                        0.0              50.0   50.0                                                  0.1              45.0   55.0                                                  4.6              36.0   64.0                                                  4.7               0.0   100.0                                                 5.2               0.0   100.0                                                 5.3              50.0   50.0                                                  7.8              50.0   50.0                                                  ______________________________________                                    

The Lambda sequence has been published by O'Conner et al. inBiophys.J.74:A285 (1998) and by Garner, et al., at the MutationDetection 97 4th International Workshop, Human Genome Organization, May29-Jun. 2, 1997, Brno, Czech Republic, Poster no. 29. The 100 bp Lambdafragment sequence (base positions 32011-32110) was used as a standard(available from FMC Corp. available from FMC Corp. BioProducts,Rockland, Me.). The mutation was at position 32061. The chart belowlists the primers used:

    ______________________________________                                                Primers                                                               ______________________________________                                                Forward Primer:                                                               5'-GGATAATGTCCGGTGTCATG-3'                                                    Reverse Primer:                                                               3'-GGACACAGTCAAGACTGCTA-5'                                            ______________________________________                                    

FIG. 21 is a chromatogram of the wild type strand analyzed under theabove conditions. The peak appearing has a retention time of 4.78minutes and an area of 98621.

FIG. 22 is the Lambda mutation analyzed in identical conditions as FIG.21 above. Two peaks are apparent in this chromatogram, with retentiontimes of 4.32 and 4.68 minutes and a total area of 151246.

The Mutation Separation Factor is calculated by applying these variouspeak areas to the above MSF equation. Thus, using the definition statedhereinabove, MSF=(area peak 2-area peak 1)/area peak 1, the MSF would be(151246-98621)/98621, or 0.533.

EXAMPLE 14 Effect of multivalent cation decontamination measures onsample resolution by DMIPC

The separation shown in FIG. 18 was obtained using a WAVE™ DNA FragmentAnalysis System (Transgenomic, Inc., San Jose, Calif.) under thefollowing conditions: Column: 50×4.6 mm i.d. containing alkylatedpoly(styrene-divinylbenzene) beads (DNASep®, Transgenomic, Inc.); mobilephase 0.1 M TEAA (1 M concentrate available from Transgenomic, Inc.)(Eluent A), pH 7.3; gradient: 50-53% 0.1 M TEAA and 25.0% acetonitrile(Eluent B) in 0.5 min; 53-60% B in 7 min; 60-100% B in 1.5 min; 100-50%B in 1 min; 50% B for 2 min. The flow rate was 0.9 mL/min, UV detectionwas at 254 nm, and the column temperature was 56° C. The sample was 2 μL(=0.2 μg DNA, DYS271 209 bp mutation standard with an A to G mutation atposition 168).

FIG. 19 is the same separation as performed in FIG. 18, but afterchanging the guard cartridge (20×4.0 mm, chelating cartridge, part no.530012 from Transgenomic, Inc.) and replacing the pump-valve filter(Part no. 638-1423, Transgenomic, Inc.). The guard cartridge haddimensions of 10×3.2 mm, containing iminodiacetate chelating resin of2.5 mequiv/g capacity and 10 μm particle size, and was positioneddirectly in front of the injection valve.

FIG. 20 is the same separation as performed in FIG. 19, but afterflushing the column for 45 minutes with 0.1 M TEAA, 25% acetonitrile,and 32 mM EDTA, at 75° C.

EXAMPLE 15 Hybridization of mutant and wild type DNA fragments

A mixture of two homoduplexes and two heteroduplexes was produced by ahybridization process. In this process, a DYS271 209 bp mutationstandard containing a mixture of the homozygous mutant DNA fragment(with an A to G mutation at position 168) combined with thecorresponding wild type fragment in an approximately 1:1 ratio (themixture is available as a Mutation Standard from Transgenomic, Inc., SanJose, Calif.; the mutation is described by Seielstad et al., Human Mol.Genet. 3:2159 (1994)) was heated at 95° C. for 3-5 minutes then cooledto 25° C. over 45 minutes. The hybridization process is shownschematically in FIG. 17.

EXAMPLE 16 Alkylation of Poly(Styrene-Divinylbenzene) Polymer Beads

The following procedures were carried out under nitrogen (Air Products,Ultra Pure grade, Allentown, Pa.) at a flow rate of 250-300 mL/min. 25 gof the beads prepared in Example 1 were suspended in 150-160 g of1-chlorooctadecane (product no. 0235, TCI America, Portland, Oreg.)using a bow shaped mixer (use a 250 mL wide neck Erlenmeyer flask). Thetemperature was set to 50-60° C. to prevent the 1-chlorooctadecane fromsolidifying. Larger pieces of polymer were broken up to facilitatesuspending. The solution was mixed using a stirrer (Model RZRI, Caframo,ONT NOH2T0, Canada) with the speed set at 2. The polymer suspension wastransferred into a three neck bottle (with reflux condenser, overheadstirrer and gas inlet). 52-62 g of 1-chlorooctadecane were used to rinsethe Erlenmeyer flask and were added to the three neck bottle. The bottlewas heated in an ethylene glycol bath set at 80° C. The solution wasmixed using a stirrer (Caframo) with the speed set at 0. After 20minutes, the reaction was started by addition of 1.1 g AlCl₃ powder(product no. 06218, Fluka, Milwaukee, Wis.) and continued for 16-18 h.

After the reaction, the polymer was separated from excess 1-chlorooctadecane by centrifugation followed by consecutive washingsteps:

    ______________________________________                                        Addition           Comment                                                    ______________________________________                                        50 mL conc. HCl, 50-60 mL n-heptane                                                              4 repetitions, with recycled                                                  heptane                                                    100 mL H.sub.2 O, 50-60 mL n-heptane                                                             1 repetition, with fresh heptane                           50 mL conc. HCl, 50-60 mL n-heptane                                                              1 repetition, with fresh heptane                           100 mL H.sub.2 O, 50-60 mL n-heptane                                                             1 repetition, fresh heptane                                150 mL H.sub.2 O, no n-heptane                                                                   3 repetitions, use plastic stirrer                                            to break up chuncks of polymer                                                beads. Repeat steps 4 and 5                                                   three times. Shake for two                                                    minutes with no centrifugation.                            100 mL THF         3 repetitions                                              100 mL THF/n-heptane                                                                             1 repetition                                               100 mL n-heptane   1 repetition                                               100 mL THF         1 repetition                                               100 mL CH.sub.3 OH 4 repetitions                                              ______________________________________                                    

In the steps where aqueous solvents (HCl or H₂ O) were used, the polymerwas shaken for 30 seconds with the aqueous phase before addingn-heptane. n-Heptane was then added and the mixture was shakenvigorously for 2 min.

After the final polymeric beads were dried at 40-50° C. for 2-3 hr itwas ready for packing.

EXAMPLE 17 Column packing procedure

After weighing out 1.2 grams of oven dried polymeric beads, form aslurry with 10 mL tetrahydrofuran (THF) and place in a sonicator under afume hood for 15 min. The add 5 mL of THF and 5 mL of methanol (MeOH)and sonicate an additional 10 min. Pre-fill a packing assembly with 20mL MeOH. Pour the slurry slowly into the packing assembly. Turn on aHaskel pump (Haskel International, Inc., Burbank, Calif.) and slowlyincrease packing pressure to 5000 psi for the initial packing phase.After 10 min, slowly increase packing pressure to 9000 psi and set thesecondary packing phase for 20 min. After 20 min, change the packingeluent from MeOH to 0.05 M Na₄ EDTA. The set the final packing phase for40 min.

While the foregoing has presented specific embodiments of the presentinvention, it is to be understood that these embodiments have beenpresented by way of example only. It is expected that others willperceive and practice variations which, though differing from theforegoing, do not depart from the spirit and scope of the invention asdescribed and claimed herein.

The invention claimed is:
 1. A method for separating a mixture of doublestranded polynucleotide fragments having up to 1500 base pairs,comprisinga) flowing the mixture through a separation column containingpolymer beads having an average diameter of 0.5 to 100 microns, saidbeads being unsubstituted polymer beads or polymer beads substitutedwith a moiety selected from the group consisting of methyl, ethyl, orhydrocarbon having from 23 to 1,000,000 carbons, wherein said beads arecharacterized by being substantially free from multivalent cations whichare free to bind with DNA and having a DNA Separation Factor of at least0.5, and b) separating said mixture of polynucleotides.
 2. The method ofclaim 1 wherein the beads have been subjected to acid wash treatment toremove any residual surface metal contaminants.
 3. The method of claim 1wherein the beads have been subjected to treatment with multivalentcation binding agent.
 4. A method of claim 1, including eluting saidmixture with a mobile phase comprising a counterion agent and an organicsolvent, wherein said organic solvent is water soluble.
 5. A method ofclaim 4, wherein said solvent is selected from the group consisting ofalcohol, nitrile, dimethylformamide, tetrahydrofuran, ester, ether, andmixtures of one or more thereof.
 6. A method of claim 4 wherein saidcounterion agent is selected from the group consisting of lower alkylprimary amine, lower alkyl secondary amine, lower alkyl tertiary amine,lower trialkylammonium salt, quaternary ammonium salt, and mixtures ofone or more thereof.
 7. A method of claim 4 wherein said counterionagent is selected from the group consisting of octylammonium acetate,octadimethylammonium acetate, decylammonium acetate, octadecylammoniumacetate, pyridiniumammonium acetate, cyclohexylammonium acetate,diethylammonium acetate, propylethylammonium acetate,propyldiethylammonium acetate, butylethylammonium acetate,methylhexylammonium acetate, tetramethylammonium acetate,tetraethylammonium acetate, tetrapropylammonium acetate,tetrabutylammonium acetate, dimethydiethylammonium acetate,triethylammonium acetate, tripropylammonium acetate, tributylammoniumacetate, tetrapropylammonium acetate, tetrabutylammonium acetate,triethylammonium hexafluoroisopropyl alcohol, and mixtures of one ormore thereof.
 8. A method of claim 4 wherein said counterion agentincludes an anion, said anion is selected from the group comprisingacetate, carbonate, phosphate, sulfate, nitrate, propionate, formate,chloride, and bromide.
 9. A method of claim 1 wherein said separation ismade by Matched Ion Polynucleotide Chromatography.